Our research focuses on elucidating new fundamental principles governing T cell activation, regulation, and effector function, and employing these to develop more effective vaccine and immunotherapy strategies for HIV and cancer. This involves several steps that together comprise a push-pull approach. First, we optimize the antigen to improve immunogenicity by epitope enhancement, changing the amino acid sequence to increase affinity for the relevant major histocompatibility (MHC) molecule. We have done this for several cancer antigens, including the prostate cancer antigen, TARP, and have now opened a phase I/II clinical trial in prostate cancer patients with rising PSA levels to determine whether the TARP peptide vaccine can reduce the rate of PSA rise. We have also enhanced a peptide from HIV reverse transcriptase that is mutated in response to lamivudine, and we are collaborating on a clinical trial with Dr. Robert Yarchoan (NCI) to determine whether this vaccine can provide selective pressure against outgrowth of drug-resistant mutant HIV. The second step is to push the response with molecular adjuvants, such as cytokines and Toll-like receptor (TLR) ligands, to improve not only the quantity but also the quality of the response. We published this year that IL-15 is an important mediator of CD4 T cell help for CD8 T cells, in that it is sufficient to substitute for help in animals depleted of CD4 T cells, to allow a memory CD8 response and prevent TRAIL-mediated apoptosis, and also it is necessary for help. If dendritic cells (DCs) cannot be induced by helper cells to make IL-15, then the help is not adequately delivered to CD8 T cells. We also previously found that IL-15 increased the avidity of the CD8 T cells, necessary for effective clearance of virus or tumor cells. We also found that IL-15 induces a novel set of T cells expressing only the CD8&amp;#945;chain, not the &amp;#946;chain, that are distinct from conventional CD8 T cells and distinct from the CD8&amp;#945;-only T cells found in the gut intraepithelial compartment. These cells make substantial levels of interferon-&amp;#947;and have lytic activity. We also investigated TLR ligands as adjuvants, because these can mature DCs and induce their production of cytokines like IL-12 and IL-15. We published a study showing synergy between pairs of TLR ligands that work through different intracellular signal transducers, MyD88 or TRIF, and determined the mechanism in DCs involving unidirectional cross-talk from TRIF to enhance MyD88-dependent cytokine production. We have now found a triple TLR ligand combination that induces more effective protection against virus infection. This combination does not increase T cell quantity, but improves quality by inducing higher avidity T cells, and induces more IL-15 production, accounting for the higher avidity. We tested the triple TLR ligands, IL-15, both or neither as vaccine adjuvants in a peptide-prime, MVA-boost mucosal vaccine for SIV in macaques, challenging intrarectally with SIVmac251. Only the macaques receiving both types of adjuvants showed some protection, so we investigated correlates of protection. In the adaptive immune arm, surprisingly only polyfunctional CD8 T cells specific for SIV antigens, but not total specific T cells measured by peptide-MHC tetramer binding, correlated with protection. In the innate immune arm, we found the adjuvants induced long-lived innate protection by APOBEC3G. Thus, vaccine strategies that induce both innate and adaptive immunity may be the most efficacious. The third step is to target the immune response to the relevant tissue, the mucosa in the case of HIV. We have studied mucosal T cell trafficking and discovered a lack of equilibrium between T cells populating the intraepithelial compartment and the lamina propria in the small intestine, leading to a distinct founder effect causing a narrower repertoire of intraepithelial T cells. We also found that homing to the large intestine is governed in part by DCs from colon patches, using a mechanism distinct from that in the small intestine. We are trying to develop approaches to selectively target T cells to the large intestine. We also found that one can prime mucosally with a DNA vaccine and direct a response to mucosa after boosting systemically with a viral vector vaccine and vice versa, allowing for new combinations of vaccines to induce both systemic and mucosal immunity. The fourth step is to pull the response by removing the brakes, i.e., blocking the negative regulatory mechanisms that inhibit the immune response. We previously discovered a new immunoregulatory pathway involving NKT cells that suppresses tumor immunity. The NKT cells make IL-13 that induces myeloid cells to make TGF-&amp;#946;, which suppresses the CD8 T cell response. However, NKT cells can also protect against tumors, so we needed to resolve this paradox. We found that type I NKT cells (using an invariant TCR&amp;#945;chain) protected, whereas type II NKT cells (using diverse T cell receptors) suppressed immunity. Moreover, selective activation of type I or type II NKT cells showed they cross-regulated each other, defining a new immunoregulatory axis, analogous to the axis between Th1 and Th2 cells that has profoundly affected immunology. The balance along the NKT axis could influence subsequent adaptive immune responses. We found that type II NKT cells also suppress conventional CD4 and CD8 antigen-specific T cells, so they are broadly suppressive. We are examining the mechanisms of suppression and also investigating the relationship between suppressive NKT cells and CD25+ Foxp3+ T regulatory cells. In trying to tip the balance along the NKT regulatory axis, we find that blocking IL-13 can delay growth of spontaneous, autochthonous tumors even in HER-2 transgenic mice that develop aggressive independent tumors in all 10 mammary glands. Conversely, stimulating with a type I NKT cell agonist can protect against tumors. To expand on potential NKT agonists beyond the classic ones, we examined a &amp;#946;-mannosyl analog of &amp;#945;-galactosylceramide, and found that in contrast to other &amp;#946;-linked sugar glycolipids, this one is also protective. However, its mechanism of protection is different from that of the classic &amp;#945;-GalCer. Thus, using different NKT agonists, one may be able to tailor the protective response to fit different conditions and to achieve synergy between different mechanisms of protection. A key mediator of the NKT regulatory pathway and an important regulator of T regulatory cells is TGF-&amp;#946;. We have found that blockade of TGF-&amp;#946;can protect against certain tumors in mice, and can synergize with anti-cancer vaccines in two mouse models. The protection is dependent on CD8 T cells, and when used in combination with a vaccine, the anti-TGF-&amp;#946;increases the number of both total and high avidity CD8 T cells. We have translated this into a human clinical trial of a human anti-TGF-&amp;#946;monoclonal antibody in a CRADA with Genzyme, in melanoma and renal cell cancer. The phase I study showed some activity (one long partial remission, 2 mixed responses and 2 cases of stable disease among 22 patients). We are now developing phase II clinical trials in melanoma and prostate cancer, and would like to combine the antibody with a vaccine to induce anti-cancer immunity. Finally, we recently published that an adenovirus vaccine expressing the extracellular and transmembrane domains of HER-2 [summary truncated at 7800 characters]